Effect of Lipids on Vascular Graft Healing

项目总结/摘要 内皮细胞（EC）迁移对于动脉损伤（如球囊扩张期间发生的损伤）的愈合至关重要 血管成形术或旁路移植术。氧化低密度脂蛋白（oxLDL）和溶血磷脂酰胆碱（lysoPC）， LDL氧化的产物和oxLDL中的主要溶血磷脂，在动脉粥样硬化动脉中积累， 在动脉损伤部位，并抑制EC迁移。有限的EC愈合导致血栓形成，光滑 肌肉细胞增殖和最终的再狭窄。已证明EC快速愈合可限制内膜增生， 血管成形术或假体移植物植入后的SIA。 脂质氧化产物导致细胞内游离钙离子浓度（[Ca 2 +]i）的长期升高， 抑制EC迁移。我们发现oxLDL和lysoPC激活经典瞬时受体电位6 （TRPC 6）通道，这引发了一系列事件，导致TRPC 5的激活，负责 [Ca 2 +]i的延长增加抑制EC迁移。TRPC 6对TRPC 5激活的重要性 体内级联反应表现为：1）高胆固醇饮食激活TRPC 6和TRPC 5，2）抑制TRPC 6和TRPC 5的表达。 在高胆固醇饮食的野生型小鼠中，动脉愈合的抑制作用，但在TRPC 6-/- 或TRPC 5-/-小鼠。不幸的是，尚未开发出用于临床使用的TRPC 6抑制剂。我们发现， 然而，TRPC 6激活需要钙通过花生四烯酸调节钙（ARC）流入， 渠道基于我们的初步数据，我们假设脂质氧化产物激活TRPC 6通道， 通过激活磷脂酶A2（PLA 2）释放细胞花生四烯酸，打开ARC通道。的 通过ARC通道进入的Ca 2+激活Src激酶，导致TRPC 6通道开放。 nels。为了验证这一假设，我们将确定初始lysoPC诱导的[Ca 2 +]i增加的机制， 研究PLA 2激活和ARC通道的作用以及所涉及的PLA 2的特定类型。我们还将 探索[Ca 2 +]i增加激活TRPC 6的机制，研究特异性Src激酶 它被激活了。PLA 2和Src激酶抑制剂对TRPC 6活化和EC迁移的影响 将研究这些抑制剂阻断TRPC活化并促进EC愈合和抑制 将使用小鼠颈动脉损伤模型在进食食物或高脂肪饮食的动物中探索内膜增生 和兔血管移植物模型。 拟议的研究将导致更好地了解脂质氧化的机制， 抑制EC迁移并促进内膜增生。这将有助于制定有针对性的干预措施 抑制TRPC 6活化并促进EC迁移到血管成形术部位或心血管植入物上， 改善心血管疾病患者血管介入治疗后的结局。

Project Summary/Abstract Endothelial cell (EC) migration is essential to heal arterial injuries such as those that occur during balloon angioplasty or bypass grafting. Oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC), a product of LDL oxidation and the major lysophospholipid in oxLDL, accumulate in atherosclerotic arteries and at sites of arterial injury, and inhibit EC migration. Limited EC healing contributes to thrombogenicity, smooth muscle cell proliferation, and eventual restenosis. Rapid EC healing has been shown to limit intimal hyperpla- sia after angioplasty or prosthetic graft implantation. Lipid oxidation products cause a prolonged rise in intracellular free calcium ion concentration ([Ca2+]i) that inhibits EC migration. We discovered that oxLDL and lysoPC activate canonical transient receptor potential 6 (TRPC6) channels, and this initiates a series of events leading to activation of TRPC5 that is responsible for the prolonged increase in [Ca2+]i that inhibits EC migration. The importance of the TRPC6 to TRPC5 activation cascade in vivo is shown by: 1) the activation of TRPC6 and TRPC5 by a high cholesterol diet, and 2) the inhi- bition of arterial healing in wild-type mice on a high cholesterol diet, but dramatically less inhibition in TRPC6-/- or TRPC5-/- mice. Unfortunately, no TRPC6 inhibitor has been developed for clinical use. We have discovered, however, that TRPC6 activation requires influx of calcium through arachidonic acid-regulated calcium (ARC) channels. Based on our preliminary data, we postulate that lipid oxidation products activate TRPC6 channels by activating phospholipase A2 (PLA2) to release cellular arachidonic acid that opens ARC channels. The Ca2+ that enters through the ARC channels activates a Src kinase that leads to the opening of TRPC6 chan- nels. To test this hypothesis we will determine the mechanism of the initial lysoPC-induced increase in [Ca2+]i, investigating the role of PLA2 activation and ARC channels and the specific type of PLA2 involved. We will also explore the mechanism by which the increase in [Ca2+]i activates TRPC6, investigating the specific Src kinase that is activated. The effect of PLA2 and Src kinase inhibitors on TRPC6 activation and EC migration in vitro will be studied, and the ability of these inhibitors to block TRPC activation and promote EC healing and inhibit intimal hyperplasia will be explored in animals on a chow or high fat diet using a mouse carotid injury model and a rabbit vascular graft model. The proposed studies will lead to a better understanding of the mechanisms by which lipid oxidation prod- ucts inhibit EC migration and promote intimal hyperplasia. This will allow development of targeted interventions to inhibit TRPC6 activation and promote EC migration into angioplasty sites or onto cardiovascular implants to improve the outcome after vascular interventions in patients with cardiovascular disease.